Developmental stage determines efficiency of gene transfer to muscle satellite cells by in utero delivery of adeno-associated virus vector serotype 2/9

Abstract

Efficient gene transfer to muscle stem cells (satellite cells) has not been achieved despite broad transduction of skeletal muscle by systemically administered adeno-associated virus serotype 2/9 (AAV-9) in mice. We hypothesized that cellular migration during fetal development would make satellite cells accessible for gene transfer following in utero intravascular injection. We injected AAV-9 encoding green fluorescent protein (GFP) marker gene into the vascular space of mice ranging in ages from post-coital day 12 (E12) to postnatal day 1 (P1). Satellite cell transduction was examined using: immunohistochemistry and confocal microscopy, satellite cell migration assay, myofiber isolation and FACS analysis. GFP positive myofibers were detected in all mature skeletal muscle groups and up to 100% of the myofibers were transduced. We saw gestational variation in cardiac and skeletal muscle expression. E16 injection resulted in 27.7 ± 10.0% expression in satellite cells, which coincides with the timing of satellite cell migration, and poor satellite cell expression before and after satellite cell migration (E12 and P1). Our results demonstrate that efficient gene expression is achieved in differentiated myofibers and satellite cells after injection of AAV-9 in utero. These findings support the potential of prenatal gene transfer for muscle based treatment strategies.

Genetic Basis for Congenital Heart Disease: Revisited: A Scientific Statement From the American Heart Association

“…432 Adeno-associated virus has proven to be an extremely efficient method for postnatal gene transfer to cardiomyocytes, and adeno-associated virus can be combined with CRISPR/Cas9 to efficiently introduce somatic mutations into cardiomyocytes. Although it is possible to deliver adeno-associated virus to late-stage mouse embryos, 433 unfortunately at the present time, adeno-associated virus transduction of mid-gestation mouse embryos and noncardiomyocytes (such as endothelial cells and valve cells) is inefficient and thus not applicable to many heart development studies.…”

Established cell surface markers efficiently isolate highly overlapping populations of skeletal muscle satellite cells by fluorescence-activated cell sorting

“…Yet, within the non-hematopoietic, non-fibroadipogenic subset of muscle mononuclear cells, many surface marker schemes have been reported to positively enrich satellite cells. Some of the cell surface antigens employed are used independently of other positive markers, including VCam1, α7-integrin, NCam1, cMet, m-Cadherin, and Synd3/4 [5, 15, 18, 21, 24, 34], and some are used in combination, including β1-integrin and CXCR4 or α7-integrin and CD34 [11, 14, 19, 29, 32, 33, 35]. However, it remains unknown if all of these surface proteins are expressed on the same satellite cells.…”

Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep

“…These studies, together with our inability to detect anti-AAV9 antibodies in the fetal serum prior to or after AAV9.GFP IUGT, make it unlikely that anti-AAV9 antibodies prevented AAV9.GFP transduction. Finally, the AAV9.GFP vector used in the current study has been demonstrated to successfully transduce mouse hepatocytes following in utero delivery ([ 19 ] and preliminary data not shown) and pulmonary epithelial cells in 1 week old sheep following endobronchial delivery [ 62 ] confirming that the vector is of good quality and capable of transducing cells. Thus, we believe, that the absence of GFP + hepatocytes following in utero AAV9.GFP delivery in the current study is secondary to the absence or low levels of the serotype’s ligand on the sheep fetal liver at the time of injection.…”